Phylogeography of hepatitis B virus: the role of Portugal in the early dissemination of HBV worldwide

Copyright: © 2022 Marcelino et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.In Portugal, the genetic diversity, origin of HBV and the Portuguese role in the dissemination of HBV worldwide were never investigated. In this work, we studied the epidemic history and transmission dynamics of HBV genotypes that are endemic in Portugal. HBV pol gene was sequenced from 130 patients followed in Lisbon. HBV genotype A was the most prevalent (n = 54, 41.5%), followed by D (n = 44, 33.8%), and E (n = 32, 24.6%). Spatio-temporal evolutionary dynamics was reconstructed in BEAST using a Bayesian Markov Chain Monte Carlo method, with a GTR nucleotide substitution model, an uncorrelated lognormal relaxed molecular clock model, a Bayesian skyline plot, and a continuous diffusion model. HBV subgenotype D4 was the first to be introduced in Portugal around 1857 (HPD 95% 1699-1931) followed by D3 and A2 a few decades later. HBV genotype E and subgenotype A1 were introduced in Portugal later, almost simultaneously. Our results indicate a very important role of Portugal in the exportation of subgenotypes D4 and A2 to Brazil and Cape Verde, respectively, in the beginning of the XX century. This work clarifies the epidemiological history of HBV in Portugal and provides new insights in the early and global epidemic history of this virus.This work was performed in the context of Rute Marcelino PhD study, whose student’s fellowship (SFRH/BD/99507/2014) was supported by the Portuguese Agency for Scientific Research, Fundação para a Ciência e Tecnologia (FCT), POCH program, Portugal 2020, and European Union/Social European Fund (FSE). This work was also supported by FCT through funds of AA's projects GHTM-UID/Multi/04413/2013 and GHTM-UID/04413/2020 and also NT's projects UIDB/04138/2020 and UIDP/04138/2020.info:eu-repo/semantics/publishedVersio

Alpha2-Containing Glycine Receptors Promote Neonatal Spontaneous Activity of Striatal Medium Spiny Neurons and Support Maturation of Glutamatergic Inputs

Glycine receptors (GlyRs) containing the α2 subunit are highly expressed in the developing brain, where they regulate neuronal migration and maturation, promote spontaneous network activity and subsequent development of synaptic connections. Mutations in GLRA2 are associated with autism spectrum disorder, but the underlying pathophysiology is not described yet. Here, using Glra2-knockout mice, we found a GlyR-dependent effect on neonatal spontaneous activity of dorsal striatum medium spiny neurons (MSNs) and maturation of the incoming glutamatergic innervation. Our data demonstrate that functional GlyRs are highly expressed in MSNs of one-week-old mice, but they do not generate endogenous chloride-mediated tonic or phasic current. Despite of that, knocking out the Glra2 severely affects the shape of action potentials and impairs spontaneous activity and the frequency of miniature AMPA receptor-mediated currents in MSNs. This reduction in spontaneous activity and glutamatergic signaling can attribute to the observed changes in neonatal behavioral phenotypes as seen in ultrasonic vocalizations and righting reflex. In adult Glra2-knockout animals, the glutamatergic synapses in MSNs remain functionally underdeveloped. The number of glutamatergic synapses and release probability at presynaptic site remain unaffected, but the amount of postsynaptic AMPA receptors is decreased. This deficit is a consequence of impaired development of the neuronal circuitry since acute inhibition of GlyRs by strychnine in adult MSNs does not affect the properties of glutamatergic synapses. Altogether, these results demonstrate that GlyR-mediated signaling supports neonatal spontaneous MSN activity and, in consequence, promotes the functional maturation of glutamatergic synapses on MSNs. The described mechanism might shed light on the pathophysiological mechanisms in GLRA2-linked autism spectrum disorder cases

Control of protein synthesis and memory by GluN3A-NMDA receptors through inhibition of GIT1/mTORC1 assembly

De novo protein synthesis is required for synapse modifications underlying stable memory encoding. Yet neurons are highly compartmentalized cells and how protein synthesis can be regulated at the synapse level is unknown. Here, we characterize neuronal signaling complexes formed by the postsynaptic scaffold GIT1, the mechanistic target of rapamycin (mTOR) kinase, and Raptor that couple synaptic stimuli to mTOR-dependent protein synthesis; and identify NMDA receptors containing GluN3A subunits as key negative regulators of GIT1 binding to mTOR. Disruption of GIT1/mTOR complexes by enhancing GluN3A expression or silencing GIT1 inhibits synaptic mTOR activation and restricts the mTOR-dependent translation of specific activity-regulated mRNAs. Conversely, GluN3A removal enables complex formation, potentiates mTOR-dependent protein synthesis, and facilitates the consolidation of associative and spatial memories in mice. The memory enhancement becomes evident with light or spaced training, can be achieved by selectively deleting GluN3A from excitatory neurons during adulthood, and does not compromise other aspects of cognition such as memory flexibility or extinction. Our findings provide mechanistic insight into synaptic translational control and reveal a potentially selective target for cognitive enhancement

Efectos de los pesticidas organoclorados sobre la neurotransmisión glutamatérgica en cultivos primarios neuronales. Interacciones con el sistema neuroendocrino.

Los pesticidas organoclorados (POCs) son compuestos altamente persistentes en el medio ambiente y dada su capacidad de acumularse en los tejidos grasos animales se biomagnifican a lo largo de la cadena trófica. Estos contaminantes ambientales son antagonistas del receptor GABAA, lo que les confiere sus propiedades convulsivas tras una intoxicación aguda. En este estudio hemos planteado la hipótesis de que la exposición prolongada al pesticida dieldrín en cultivos primarios de neuronas corticales podría alterar la funcionalidad, expresión o localización de los receptores de glutamato como consecuencia del bloqueo mantenido del receptor GABAA. La exposición durante 6 días in vitro a una concentración no citotóxica (60nM) de dieldrín produjo la internalización de los receptores NMDA que contienen la subunidad NR2B (NR1/NR2B), pero no NR2A, y disminuyó también los niveles del receptor metabotrópico de glutamato 5 (mGluR5). El tratamiento con los neuroesteroides estradiol o alopregnanolona previno la reducción de los receptores NR1/NR2B en la membrana inducida por el dieldrín. Además, la exposición prolongada a 200nM dieldrin disminuyó la expresión génica de NR2A, efecto que fue revertido sólo por alopregnanolona. En neuronas corticales maduras, el dieldrín causó el truncamiento de NR2B mediado por las calpaínas y redujo la interacción de ésta subunidad con SAP102, efecto de nuevo prevenido por alopregnanolona. A pesar de que ambas hormonas restauraron la funcionalidad del receptor NMDA, alopregnanolona (pero no estradiol) revirtió tanto la inhibición del receptor GABAA como la muerte neuronal causadas por el dieldrín. Posteriormente, estudiamos los efectos de dieldrín, endosulfán y lindano sobre los receptores de estrógenos (ER) en cultivos primarios de neuronas corticales y de células granulares de cerebelo (CGC). Todos ellos inhibieron la unión de [3H]-estradiol en ambos tipos neuronales, siendo el dieldrín en CGC aquel con mayor afinidad sobre el ER. Además, dieldrín y endosulfán causaron un aumento de la fosforilación de Akt en neuronas corticales, efecto que fue inhibido por el antagonista de ERβ, PHTPP. En cambio, la fosforilación de Akt y ERK1/2 inducida por dieldrín en CGC fue mediada por la activación múltiple de ERα, ERβ, y GPR30. El lindano inhibió el efecto del estradiol sobre estas kinasas. En neuronas corticales, los tres pesticidas activaron ERK1/2 a través de los receptores GABAA y de glutamato. La exposición a largo plazo a los POCs redujo los niveles de ERα pero no de ERβ. Finalmente, comparamos la neurotoxicidad de los POCs en CGC y neuronas corticales. El dieldrín y el endosulfán fueron más tóxicos que el lindano en ambos cultivos. Se observó también que las CGC maduras son más sensibles que las neuronas corticales a la neurotoxicidad inducida por los POCs. Por el contrario, las neuronas corticales inmaduras, especialmente las glutamatérgicas, fueron más susceptibles a la exposición prolongada a concentraciones bajas de estos pesticidas. Además, la fosforilación de Akt y ERK1/2 se vio disminuida tras 24 horas de exposición a dieldrín o endosulfan en CGC. El tratamiento con estradiol e insulina previno la muerte apoptótica mediada por caspasa-3 causada por estos pesticidas a través de la activación conjunta de ERK1/2 y Akt. Este trabajo confirma que el bloqueo permanente del receptor GABAA por dieldrín provoca una reducción de la neurotransmisión glutamatérgica, lo que podría afectar a funciones cerebrales como la memoria y el aprendizaje. Además, los POCs son capaces de mimetizar o inhibir algunas de las acciones no genómicas del estradiol, lo que puede interferir con la señalización fisiológica de esta hormona en el cerebro. Este estudio revela también la susceptibilidad específica de las neuronas glutamatérgicas frente a la exposición prolongada a estos contaminantes y desvela los mecanismos moleculares por los cuales los POCs producen la muerte de estas neuronas

Rearing system and behavioural adaptation of laying hens to furnished cages Sistema de cria e adaptação comportamental de poedeiras às gaiolas enriquecidas

The influences of floor and cage-rearing on behavioural adaptation to furnished cages were investigated in laying hens. Two groups of 180 Isa Brown commercial layer pullets were reared in cages (CR) or floor pens (FR) and transferred to furnished cages, where their behavioural adaptation was observed throughout the laying period (18-78 wks of age). At 17 weeks of age, hens were placed in one of the 36 furnished cages with 10 birds in each cage, each containing a nest box, perches, a dust bath, and abrasive strips. At 50 and 54 weeks of age, direct visual observation and video recording were used to assess hen’s behaviour. From 21 weeks of age, the dust-bath activities of leg-banded hens in eight cages per treatment were recorded. The amount of time spent walking was higher (3.5% vs. 1.8%) and perching was lower (7.5% vs. 13.4%) in CR hens than in FR hens (PNeste trabalho, foi avaliada a influência dos sistemas de cria (em piso e em baterias) sobre a adaptação comportamental de poedeiras em gaiolas enriquecidas. Dois grupos de 180 frangas Isa brown foram criadas em baterias (CR) ou em piso (FR) e foram transferidas para gaiolas enriquecidas, onde a adaptação comportamental foi observada durante um ciclo completo de postura (18-78 semanas de idade). Com 17 semanas de idade, as frangas foram alojadas em 36 gaiolas enriquecidas, 10 aves por gaiolas, cada uma contendo um ninho, poleiros, banho de areia e lixas de unha. Nas semanas 50 e 54, foi utilizada observação direta e imagens de vídeo para a análise do comportamento. A partir de 21 semanas de idade, a atividade de banho de areia em galinhas marcadas com anilhas foi registrada em 8 gaiolas por tratamento. As aves CR gastaram mais tempo caminhando (3,5% vs. 1,8%,) e menos tempo empoleiradas (7,5% vs. 13,4%), se comparadas com as FR (P<0,05). O número de aves presentes nos banhos de areia aumentou de 9,2% às 21 semanas para 21,4% às 72 semanas de idade, enquanto que a proporção de galinhas realizando o banho aumentou de 4,7% para 21,0% (P<0,05). Com 72 semanas de idade, todos os parâmetros de uso de banho de areia foram maiores (P<0,05) nas FR em comparação com a CR

Floor versus cage rearing: effects on production, egg quality and physical condition of laying hens housed in furnished cages Cria em piso versus cria em bateria: efeitos na produção, qualidade de ovos e condição física de poedeiras alojadas em gaiolas enriquecidas

The influences of floor- and cage-rearing on egg production, egg quality and physical condition were investigated in laying hens housed in furnished cages. Two groups of 180 Isa Brown commercial layer pullets were reared in cages (CR) or floor pens (FR) and transferred to furnished cages, where their production, egg quality and physical condition was observed throughout the laying period (18-78wks of age). At 17 weeks of age, hens were placed in one of 36 furnished cages with 10 birds in each cage, each containing a nest box, perches, a dust bath, and abrasive strips. From 19 to 78 weeks of age, egg production data were collected daily. Commercial egg quality was assessed monthly. At, 19 and 78 weeks of age, claw length and feather cover were visually assessed using a four-point scale in a sample (10%) of hens. Production variables were above breeders’ standards and not significantly affected by rearing system. Dirty eggs and cracked eggs were more frequent in FR birds. Meat spots were significantly more frequent in FR hens at middle lay, but less frequently at the end of the laying period. Rearing system did not influence egg and yolk weight or unit Haugh and shell colour. Among FR hens, eggshell density, thickness and mass were significantly lower at the end of the laying period. Rearing system did not affect claw length, but the plumage of FR hens was negatively affected at the end of production cycle.Avaliou-se a influência dos sistemas de criação (em piso ou em baterias) sobre o desempenho produtivo, a qualidade de ovos e a condição física de poedeiras alojadas em gaiolas enriquecidas. Dois grupos de 180 frangas Isa brown foram criados em baterias (CR) ou em piso (FR) e transferidos para gaiolas enriquecidas, onde a produção, a qualidade de ovos e a condição física foram observadas durante um ciclo completo de postura (18-78 semanas de idade). Com 17 semanas de idade, as frangas foram alojadas em 36 gaiolas enriquecidas, 10 aves por gaiolas, cada uma contendo um ninho, poleiros, banho de areia e lixas de unhas. De 19 a 78 semanas de idade, a produção de ovos foi registrada diariamente. A qualidade comercial dos ovos foi medida mensalmente. Nas semanas 19 e 78 de idade, o comprimento das unhas e a condição da plumagem foram avaliadas utilizando-se uma escala de quatro pontos, numa amostra de 10% das aves. As variáveis produtivas estiveram acima dos padrões da linhagem e não foram afetadas significativamente pelo sistema de cria. Os ovos sujos ou trincados foram mais frequentes em poedeiras criadas em piso durante a metade do ciclo, mas menos frequentes ao final do ciclo de postura. O sistema de cria não influenciou o peso dos ovos, a gema, a cor da casca ou as unidades Haugh. A densidade, espessura da casca e massa de ovos foram significativamente menores em galinhas criadas em piso, ao final do ciclo de postura. O sistema de cria não afetou o comprimento das unhas, mas a conservação da plumagem das galinhas criadas em piso foi negativamente afetada ao final do ciclo de postura